The circadian clock gene bmal1 is necessary for co-ordinated circatidal rhythms in the marine isopod Eurydice pulchra (Leach).

Circadian clocks in terrestrial animals are encoded by molecular feedback loops involving the negative regulators PERIOD, TIMELESS or CRYPTOCHROME2 and positive transcription factors CLOCK and BMAL1/CYCLE. The molecular basis of circatidal (~12.4 hour) or other lunar-mediated cycles (~15 day, ~29 day), widely expressed in coastal organisms, is unknown. Disrupting circadian clockworks does not appear to affect lunar-based rhythms in several organisms that inhabit the shoreline suggesting a molecular independence of the two cycles. Nevertheless, pharmacological inhibition of casein kinase 1 (CK1) that targets PERIOD stability in mammals and flies, affects both circadian and circatidal phenotypes in Eurydice pulchra (Ep), the speckled sea-louse. Here we show that these drug inhibitors of CK1 also affect the phosphorylation of EpCLK and EpBMAL1 and disrupt EpCLK-BMAL1-mediated transcription in Drosophila S2 cells, revealing a potential link between these two positive circadian regulators and circatidal behaviour. We therefore performed dsRNAi knockdown of Epbmal1 as well as the major negative regulator in Eurydice, Epcry2 in animals taken from the wild. Epcry2 and Epbmal1 knockdown disrupted Eurydice's circadian phenotypes of chromatophore dispersion, tim mRNA cycling and the circadian modulation of circatidal swimming, as expected. However, circatidal behaviour was particularly sensitive to Epbmal1 knockdown with consistent effects on the power, amplitude and rhythmicity of the circatidal swimming cycle. Thus, three Eurydice negative circadian regulators, EpCRY2, in addition to EpPER and EpTIM (from a previous study), do not appear to be required for the expression of robust circatidal behaviour, in contrast to the positive regulator EpBMAL1. We suggest a neurogenetic model whereby the positive circadian regulators EpBMAL1-CLK are shared between circadian and circatidal mechanisms in Eurydice but circatidal rhythms require a novel, as yet unknown negative regulator. Author summary: Molecular feedback loops underlie expression of circadian ~24-hour behavioural and physiological rhythms in terrestrial animals, with PERIOD, TIMELESS and CRYPTOCHROME2 representing the negative, and CLOCK and BMAL1/CYC the positive regulators, with casein kinase 1 (CK1) acting as a modulator. In coastal marine animals, the lunar-mediated ~12.4 hour circatidal rhythm represents the dominant biological cycle but its molecular basis is unknown. The available evidence indicates that circadian and circatidal mechanisms are molecularly independent yet in the speckled-sea louse, Eurydice pulchra (Ep), pharmacological manipulation of CK1 generates correlated changes in both types of cycles, suggesting a shared component. Here, using a Drosophila cell system, we show that CK1 inhibitors alter post-translational modifications of EpCLOCK-BMAL1 leading to a reduction in their transcriptional ability and suggesting that these functional changes in EpCLOCK-BMAL1 may have produced the CK1 inhibitor-mediated circatidal phenotypes. To test this directly we used dsRNAi to knockdown the expression of EpBMAL1 in adult Eurydice captured from the wild. We also downregulated the main negative regulator EpCRY2. We observe consistent disruptions to circatidal rhythms in EpBMAl1 but not EpCRY2 knockdowns suggesting that the positive circadian regulator is involved in both circadian and lunar-mediated behaviour. [ABSTRACT FROM AUTHOR]